Prostasin is a glycosylphosphatidylinositol-anchored, trypsin-like serine protease expressed on the surface of epithelial cells (Yu, Chao et al. 1994; Chen, Skinner et al. 2001). Mature prostasin is generated by a specific cleavage between Arg12 and Ile13, and consists of a 12-amino acid light chain disulfide-linked to a 299-amino acid protease domain (Yu, Chao et al. 1995). Prostasin was first purified as a soluble enzyme from human seminal fluid, suggesting that the membrane-anchored form undergoes shedding (Yu, Chao et al. 1994). The specific physiological functions and substrates of prostasin continue to be investigated. However, studies suggest important roles in regulating epithelial sodium channel (ENaC) in airway epithelia (Adachi, Kitamura et al. 2001; Donaldson, Hirsh et al. 2002; Tong, Illek et al. 2004). Using microarray technology, prostasin was detected more strongly in cancerous ovarian epithelial cells than in normal ovarian tissue. Increased prostasin serum level in patients, in combination with cancer antigen-125 (CA-125), could be a potential serum marker for ovarian cancer (Mok, Chao et al. 2001). However, prostasin expression is significantly down-regulated in high-grade or hormone-refractory prostate tumors and is lost in highly invasive human and mouse prostate cancer cell lines (Chen, Hodge et al. 2001; Takahashi, Suzuki et al. 2003). It may serve as an invasion suppressor for human prostate and breast cancers since the transfection of prostasin cDNA reduced in vitro invasiveness of prostate carcinoma cell lines (Chen, Hodge et al. 2001), as well as breast cancer cell lines (Chen and Chai 2002). A prostasin-binding protein was found in human and mouse seminal fluid and identified as protease nexin-1 (PN-11) (Chen, Skinner et al. 2001; Chen, Zhang et al. 2004), a secreted serine protease inhibitor expressed in a variety of cell types (Bouton, Richard et al. 2003; Richard, Arocas et al. 2004). PN-1 formed an SDS-, heat-stable complex with prostasin and exhibited inhibitory activity towards prostasin with unclear potency (Chen, Zhang et al. 2004). However, PN-1 was not found forming a stable complex with prostasin in the extracts of some prostasin abundant tissues such as prostate, lung and salivary glands (Chen, Skinner et al. 2001), suggesting that additional prostasin inhibitors might exist. 1Abbreviations: PN-1, protease nexin-1; HAI-1/1B, hepatocyte growth factor activator inhibitor-1 and/or 1B; CHO, Chinese hamster ovary; KD1 and KD2, N- and C-terminal Kunitz domain of HAI-1B; sHAI-1B, soluble form of HAI-1B encompassing the extracellular domain; TT buffer, 20 mM Tris.Cl, pH 9.0 containing 0.01% Triton X-100.
Hepatocyte growth factor activator inhibitor-1B (HAI-1B) is a splicing isoform of HAI-1, a bi-Kunitz type serine protease inhibitor found mainly in epithelium (Kataoka, Suganuma et al. 1999; Itoh, Yamauchi et al. 2000). HAI-1 and/or HAI-1B (referred to herein as HAI-1/1B) is thought to be involved in tissue regeneration and tumorigenesis by inhibiting the activation of pro-HGF. Enhanced expression of HAI-1 was noted in regenerating epithelial cells such as the regenerative colon epithelium of acetic acid-induced mouse colitis models (Itoh, Kataoka et al. 2000), indicating a possible role in regulating the level of HGF activation. In agreement with this hypothesis, HAI-1 expression in colorectal mucosa was down-regulated in adenocarcinoma (Kataoka, Uchino et al. 1998; Kataoka, Hamasuna et al. 2000), where pro-HGF processing is enhanced (Kataoka, Hamasuna et al. 2000). HAI-1/1B consists of an N-terminal Kunitz domain (KD1), a low density lipoprotein receptor (LDLR)-like domain, a C-terminal Kunitz domain (KD2), and transmembrane and cytoplasmic domains (Shimomura, Denda et al. 1997). KD1 is thought to be responsible for the inhibitory activity according to mutagenesis studies (Denda, Shimomura et al. 2002; Kirchhofer, Peek et al. 2003). HAI-1/1B is synthesized as a transmembrane protein on the cell surface, and appears to be subsequently secreted by shedding (Shimomura, Denda et al. 1997; Shimomura, Denda et al. 1999). The shed form of HAI-1/1B is active in inhibiting HGF activator (HGFA) (Shimomura, Denda et al. 1997) and matriptase (Lin, Anders et al. 1999), both of which are pro-HGF activators. In addition, matriptase has extracellular matrix-degrading activity and possibly plays a role in breast cancer invasion (Lin, Wang et al. 1997). Matriptase was purified from human breast milk in complex with HAI-1/1B (Lin, Anders et al. 1999), indicating that HAI-1/1B is an important regulator of matriptase. Membrane bound HAI-1 is able to complex with active HGFA. The complex is quickly released from the cell surface upon treatment with phorbol 12-myristate 13-acetate (PMA) or interleukin-1B (IL-1B) in a metalloproteinase-dependent manner (Kataoka, Shimomura et al. 2000). Since HGFA could be recovered from the complex and maintained its activity, HAI-1 may serve as a reservoir for HGFA besides being an inhibitor of it. HAI-1B has a 16-amino acid insertion after the first Kunitz domain of HAI-1 (Kirchhofer, Peek et al. 2003). No significant differences between the two splice variants have been found in terms of tissue distribution, enzymatic activity or specificity.
We identify herein physiological prostasin inhibitors based at least in part on analyzing gene expression data with the assumption that prostasin and its cognate inhibitor(s) are co-expressed. This assumption is supported by observations of co-expression of other pairs of effector molecules and their endogenous inhibitors. For example, granzyme B has been found to be expressed in the same cells as its inhibitor PI-9 (SERPINB9) in humans (Muthukumar, Ding et al. 2003) and Spi6 in mice (Phillips, Opferman et al. 2004). Co-expression of the endogenous inhibitor may serve a protective role in host cells against the proteolytic effects of the serine protease. Similarly, quantitative correlation of expression has been found between matrix metalloproteinase 1 (MMP-1) and its inhibitor TIMP-1 in myocardium (Tyagi, Kumar et al. 1995), which is thought to serve as a mechanism to regulate the activity of the MMP-1.
The expression profile of prostasin in various pathological conditions as described above, coupled with its potential role in acting as a regulator of other growth factors the dysregulation of which might underlie carcinogenesis, suggests that modulation of prostasin's interaction with other cellular factors would be an efficacious therapeutic approach. In this regard, there is a clear need to identify prostasin's physiological modulator(s). The invention fulfills this need and provides other benefits.
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